Maria Lúcia Costa de Oliveira

Fumaric aciduria: an overview and the first Brazilian case report

Fumaric aciduria is a rare metabolic disease, with 40 cases reported so far. Fumarase deficiency leads mainly to brain abnormalities, developmental delay, and great accumulation of fumaric acid in urine. This work presents the first case of fumaric aciduria described in Brazil, which presented with some interesting clinical and biochemical findings such as colpocephaly, hepatic alterations, and marked metabolic acidosis since birth. Common findings were ventriculomegaly, hypotonia, and microcephaly. Biochemically, besides the high urinary fumaric acid excretion, atypical elevation of plasma citrulline, tyrosine and methionine levels were also observed. In order to show all features and variants of fumaric aciduria, literature data of 40 patients was reviewed and compared with the case reported here. Findings in all these patients demonstrate that this disorder does not yet have its phenotype completely defined; it is important that more patients be described.

Early hematopoietic stem cell transplantation in a patient with severe mucopolysaccharidosis II: A 7 years follow-up

Mucopolysaccharidosis type II (MPS II - Hunter syndrome) is an X-linked lysosomal storage disorder caused by a deficiency in the enzyme iduronate-2 sulfatase (I2S), leading to the accumulation of the glycosaminoglycans, affecting multiple organs and systems. Enzyme replacement therapy does not cross the blood brain barrier, limiting results in neurological forms of the disease. Another option of treatment for severe MPS, hematopoietic stem cell transplantation (HSCT) has become the treatment of choice for the severe form of MPS type I, since it can preserve neurocognition when performed early in the course of the disease. To date, only few studies have examined the long-term outcomes of HSCT in patients with MPS II. We describe the seven-year follow-up of a prenatally diagnosed MPS II boy with positive family history of severe MPS form, submitted to HSCT with umbilical cord blood cells at 70 days of age. Engraftment after 30 days revealed mixed chimerism with 79% donor cells; after 7 years engraftment remains at 80%. I2S activity 30 days post-transplant was low in plasma and normal in leukocytes and the same pattern is observed to date. At age 7 years growth charts are normal and he is very healthy, although mild signs of dysostosis multiplex are present, as well as hearing loss. The neuropsychological evaluation (Wechsler Intelligence Scale for Children - Fourth Edition - WISC-IV), disclosed an IQ of 47. Despite this low measured IQ, the patient continues to show improvements in cognitive, language and motor skills, being quite functional. We believe that HSCT is a therapeutic option for MPS II patients with the severe phenotype, as it could preserve neurocognition or even halt neurodegeneration, provided strict selection criteria are followed.

Screening for hydroxyethyl starch (HES) doping in sport.

The artificial colloid hydroxyethyl starch (HES) is among the most frequently used plasma volume expanders in the medical field. However, in 1998, its misuse by the athletic community was officially reported and since 2000, HES is prohibited by the International Olympic Committee (IOC). Therefore, several methods enabling the detection of HES in urine were developed, most based on gas chromatography-mass spectrometry (GC-MS). In the present work, a simple and low-cost screening method, intended to reduce the number of samples to be analysed by GC-MS, was developed. The method is based on the acid hydrolysis of HES and detection of the resulting glucose and hydroxyethyl glucose derivatives by Benedicts reaction (reduction of copper sulfate to brick-red cuprous oxide by glucose and/or derivatives). Samples considered suspect were submitted to GC-MS analysis for identification of HES. The method was successfully applied for screening of HES in 2627 urine samples from 1346 Brazilian soccer players and 1281 athletes from the Pan-American Games (Rio de Janeiro, 2007); 71 (2.7%) samples, considered suspect, were submitted to GC-MS, but no positive results were found. Moreover, a thin layer chromatography (TLC) method was adapted for visualisation of the characteristic band pattern of HES hydrolysis products. The results indicate that the methods are efficient and useful for the screening of HES in urine.

Screening for inborn errors of metabolism in high-risk children from Rio de Janeiro, Brazil

From 1988 to 1995, our laboratory at the Institute of Chemistry of the Federal University of Rio de Janeiro, in Rio de Janeiro, screened 2650 samples from 2000 high-risk patients (mostly children) for Inborn Errors of Metabolism (IEM). Chemical tests, various chromatographic techniques and enzyme assays were performed on urine, plasma and in some cases, cerebrospinal fluid (CSF). A total of 145 cases of IEM (7.2%) was identified. These were related to: the metabolism of amino acids (41) and carbohydrates (17), organic acids (7), lysosomal enzymes (61), membrane transport system (16), metals (2), intestinal disaccharidases (1) and porphyrin metabolism (3). Furthermore, a relevant number of patients with abnormal findings is still under investigation. Biochemical results and clinical symptoms are presented and the importance of reference laboratories for the detection of IEM is stressed.

Plasma volume expanders: use in medicine and detecting misuse in sports

Plasma volume expanders comprise a heterogeneous group of substances used in medicine that are intravenously administered in cases of great blood loss owing to surgery or medical emergency. These substances, however, can also be used to artificially enhance performance of healthy athletes in sport activities, and to mask the presence of others substances. These practices are considered doping, and are therefore prohibited by the International Olympic Committee and the World Antidoping Agency. Consequently, drug testing procedures are essential. The present work provides an overview of plasma volume expanders, assembling pertinent data such as chemical characteristics, physiological aspects, adverse effects, doping and analytical detection methods, which are currently dispersed in the literature.

Systematic analysis of glycerol: colourimetric screening and gas chromatography–mass spectrometric confirmation

Glycerol is a naturally occurring polyol in the human body, essential for several metabolic processes. It is widely used in the food, pharmaceutical, and medical industries and in clinical practice as a plasma volume expander (PVE). Athletes, however, may use glycerol to mask the presence of forbidden substances or to enhance performance, inclusively through hyperhydration achieved by glycerol ingestion with added fluid. These practices are considered doping, and are prohibited by the World Anti-Doping Agency (WADA). Therefore, glycerol was introduced in the prohibited list. Doping through glycerol ingestion can readily be identified by detection of elevated glycerol concentrations in urine. In this paper, a protocol for the fast detection of glycerol in urine is proposed. It consists of a previous visual colourimetric screening, followed by a quantitative/qualitative confirmation analysis by mass spectrometry. The screening procedure involves a reaction in which polyhydric alcohols are oxidized by periodate to formic acid and formaldehyde, which is detected by the addition of a fuchsin solution. For the subsequent qualitative/quantitative confirmation analysis, a gas chromatography-mass spectrometry based approach with a non-deuterated internal standard and a drying step of only 10 min is proposed. The linear correlation was demonstrated within WADA´s threshold range. The calculated RSD were 2.1% for within-day precision and 2.8% for between-day precision. The uncertainty estimation was calculated, and a value of 2.7% was obtained. The procedure may also be used for the analysis of other polyols in urine, as for example the PVE mannitol.

Clinical and Treatment Management Decisions in Two Asymptomatic Late-Onset Pompe Disease Siblings - Further Evidence of Scoliosis as a Clinical Sentinel Sign for Juvenile Pompe Disease

disease patient was identifi ed by chance through his dermatologist due to partial alopecia. Through high CK (1,310 IU/L) and muscle/hepatic biomarkers (AST, 162 IU/L; ALT, 189 IU/L) a muscle biopsy was performed revealing vacuolated muscle pathology with H-E and PAS stains. A compound heterozygote pathogenic GAA genotype was detected (-32-13 T>G/ c.2560C>T). A series of clinical and laboratory evaluations identifi ed several abnormal clinical signs, despite the absence of clinical symptoms. As the patient had a positive DBS, diminished GAA activity in leukocytes (0.56 nml/h/mg protein – RV: 1.0–5.9), high urinary GLc4 levels on HPLC, abnormal FVC drop during sitting–supine transition (>14%), and abnormalities on his tongue and edema of his thighs, seen on MRI, the decision was taken to initiate enzymatic replacement therapy (ERT) with 20 mg/kg rhGAA (Myozyme®), even though no subjective clinical symptoms were present. After 4 years on ERT treatment and motor/respiratory rehabilitation programs, all clinical parameters worsened, especially CK levels, muscle MRI with an overall muscle substitution for fat with muscle mass volume reduction; and a higher FVC drop during sitting–supine transiti on (>18%) were noted. Based on these clinical and laboClinical and Treatment Management Decisions in Two Asymptomatic Late-Onset Pompe Disease Siblings – Further Evidence of Scoliosis as a Clinical Sentinel Sign for Juvenile Pompe Disease

Identification and Quantification of the Biomarker Glucose Tetrasaccharide Glc 4 by Thin Layer Chromatography and High Performance Liquid Chromatography in Pompe Disease Patients

Pompe disease (PD), a glycogen storage inborn error of metabolism (type II), is caused by the defi ciency of acid α-glucosidase (GAA); it can manifest itself in two forms: infantile onset (IPD) and late-onset (LOPD). Clinical presentation of this disorder is variable, depending on age at onset, level of organ involvement, progression rate, and genotype. PD is classifi ed as a glycogenosis; and, since individuals with this disorder excrete oligosaccharides in the urine, can be considered to be an oligosaccharidosis as well. Urinary tetraglucoside (Glc4), considered to be a biomarker of the disease, could be an auxiliary tool in screening for PD in suspected cases. Urine samples from 24 known patients with IPD (n = 15) and LOPD (n = 9), and normal controls (n = 215) were submitted to thin layer chromatography (TLC) analysis and high performance liquid chromatography (HPLC) quantifi Identifi cation and Quantifi cation of the Biomarker Glucose Tetrasaccharide Glc4 by Thin Layer Chromatography and High Performance Liquid Chromatography in Pompe Disease Patients

A Survey on Inborn Errors of Metabolism in 323 Neonates from the State of Rio de Janeiro, Brazil

Inborn errors of metabolism (IEM) are a relevant cause of morbidity and death among children, and neonates in particular. However, little is known about the prevalence of these disorders in Brazilian newborns. Our laboratory of IEM (LABEIM) at the Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro (UFRJ), has been working on the diagnosis of IEM since 1988. Out of 3,300 patients (90% children), screened and evaluated from 1989 to 2,000 because of a high clinical suspicion of having an IEM, 323 (9.8%) were neonates. Patients came from different regions of the state of Rio de Janeiro, in which lives approximately 8.5% of the total Brazilian population. Chemical tests, various chromatographic techniques and enzyme assays were performed in urine, plasma and in some cases, cerebrospinal fluid (CSF). This study describes our laboratory and the experience with the 323 investigated neonates, among which 28 cases (8.7%) of IEM were identified and 18 (5.6%), strongly suspected. All these cases were related mainly to the metabolism of amino acids, organic acids, lysosomal enzymes and carbohydrates. Furthermore, data on population, community and health services are presented.

Early infantile form of galactosialidosis in a female baby with a prenatal diagnosis of fetal ascites: First case in Brazil

We present the first case of an early infantile form of galactosialidosis among Brazilians. This very rare and severe lysosomal storage disease has only a dozen patients clearly diagnosed worldwide. Clinical, pathological and biochemical features were consistent with previously published findings. We detected the disorder in a 7-month-old female baby with prenatal diagnosis of ascites. Evolution of the storage disease was monitored through routine thin-layer chromatography (TLC) for urinary oligosaccharides as part of a screening program for inborn errors of metabolism (IEM) in high-risk children, carried out in Rio de Janeiro.